JP2001258581A - Use of bovine respiratory syncytial virus recombinant DNA, protein vaccines, antibodies, and transformed cells - Google Patents

Abstract

(57) [Summary] (Modified) [Problem] Bovine respiratory syncytial; B
Recombinant DNA molecules encoding viral proteins and the corresponding B derived therefrom
Provision of RS virus proteins and peptides. The above describes, in part, a number of bovine R containing F.
Based on cloning of full length cDNA encoding S virus protein. D encoding F protein
NA was inserted into vaccinia virus vectors, and these vectors were used to express the F protein in culture.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION 1. Introduction The present invention relates to a bovine RS (bovine respiratory syncytial;
(Also referred to as BRS) recombinant DNA molecules encoding viral proteins, and the corresponding BRS viral proteins and peptides derived therefrom. It is based, in part, on the cloning of full-length cDNAs encoding many bovine RS virus proteins, including F. DNA encoding F protein
Was inserted into vaccinia virus vectors, and these vectors were used to express the F protein in culture.

2. Background of the Invention 2.1. Bovine Respiratory Syncytial Virus The bovine respiratory syncytial virus strain 391-2 was isolated from a respiratory syncytial virus that spread to cattle in North Carolina from the winter of 1984 to 1985. The outbreak involved five dairy cattle, a workplace for beef calves and cows, and a workplace for beef and fattening steers. (Fetrow et al.) ., North Carolina State
University Agric. Extension Service Vet. Newsl.)
Single-stranded negative-sense RNA with envelope
The virus, the RS virus (Huang and Wertz, 198
2, J. Virol. 43: 150-157; Kingsbury et al., 1978, In
tervirology 10: 137-153) was originally isolated from chimpanzees (Morris et al., 1956, Proc. Soc. Exp.
iol. Med. 92: 544-549). Subsequently, respiratory syncytial virus was also isolated from humans, cattle, sheep and goats (Chanock
et al., 1957, Am. J. Hyg. 66: 281-290; Evermann et a
l., 1985, AM.J. Vet. Res. 46: 947-951; Lehmkuhl et
al., 1980, Arch.Virol. 65: 269-276; Lewis, FA e
tal., 1961, Med.J. Aust. 48: 932-33; Paccaud and J
acquier, 1970, Arch. Gesamte Virusforsch 30: 327-34
2). The human RS (HRS) virus is a major cause of severe lower respiratory tract infections in children during the first year of life and epidemics occur annually (Stott and Taylor, 1985, Ar
ch. Virol. 84: 1-52). Similarly, the BRS virus causes bronchiolitis and pneumonia in cattle, and epidemics of economic significance for the meat industry occur every winter (Bohlender et al.).
al., 1982, Mod.Vet.Pract. 63: 613-618; Stott and
Taylor, 1985, Arch.Virol. 84: 1-52; Stott et al.,
1980, J. Hyg. 85: 257-270). Severe disease caused by the BRS virus usually occurs most frequently in 2-4.5 month old cattle. The outbreak of the BRS virus 391-2 strain is characteristic in that many mature cows became ill, reducing milk production on one dairy by 50% and killing some animals, Young cows are mild (Fetr
ow et al., 1985, North Carolina State University A
gric. Extension Service Vet. Newsl.). The BRS virus was first isolated in 1970 (Paccaud and Jacquier,
1970, Arch. Gesamte Virusforsch. 30: 327-342) and host (Baker et al., 1986, J. Am. Vet. Med. Assoc.
189: 66-70; Castleman et al., 1985, Am. J. Vet. Re
s. 46: 554-560; Castleman et al., 1985, Am. J. Vet.
Res. 46: 547-553) and serological studies (Baker et al.,
1985, Am. J. Vet. Res. 46: 891-892; Kimman et al.,
1987, J. Clin. Microbiol. 25: 1097-1106; Stott et.
al., 1980, J. Hyg. 85: 257-270) (Van Nieuwstadt, AP et al., 1982, Pro
^{c 12 th World Congr Dis Cattle 1} :... 124-130; Verhoe
ff et al., 1984, Vet. Rec. 114: 288-293) and pathological effects. No details are given on the molecular details of the virus. One study is BRS
It only compares the proteins present in virus infected cells with those present in HRS virus infected cells (Cash et al., 1977, Virology 82: 369-379). In contrast, a detailed molecular analysis was performed on the HRS virus. A cDNA clone of the HRS virus mRNA is generated and used to identify 10 virus-specific mRNAs encoding 10 unique polypeptides;
The complete nucleotide sequence of nine of the ten genes was obtained (Collins, PL, et al., 1986, in “Concepts i
n Viral Pathogenesis II ”, Springer-Verlag., New
York; Stott and Taylor, 1985, Arch.Virol. 84: 1-5
2) HRS virus and BRS
The viruses were proposed to belong to different subgroups of respiratory syncytial virus. First, BRS and HRS viruses differ in their ability to infect various types of tissue culture cells (Paccaud and Jacquier, 1970, Arch. Gesamt.
e Virusforsch. 30: 327-342). With one exception, B
Studies show that the RS virus shows a narrower host range than the HRS virus. Matumoto et al. (1974, Arch. Ge
samte Virusforsch. 44: 280-290) reported that the NMK7 strain of BRS virus had a wider host range than the Long strain of HRS virus. Other researchers have not been able to achieve the same results with other BRS strains (Paccaud
and Jacquier, 1970, Arch. Gesamte Virusforsch. 30:
327-342; Pringle and Crass, 1978, Nature (London)
276: 501-502). A second finding that the BRS virus differs from the HRS virus came from the demonstration of antigenic differences in the major glycoprotein G of the BRS virus and the HRS virus (Orvell et al., 1987, J. Gen.
Virol. 68: 3125-3135). Studies using monoclonal antibodies have classified HRS virus strains into two subgroups based on G-glycoprotein relevance (Anderson 198).
5, J. Infect. Dis. 151: 626-633; Mufson et al., 198.
5, J. Gen. Virol. 66: 2111-2124). The G protein of the BRS virus strain included in these studies did not react with monoclonal antibodies directed against viruses from the respective HRS virus subgroup (Or
vell et al., 1987, J. Gen. Virol. 68: 3125-3135).
BRS virus is the major glycoprotein G in adhesion
, The restriction of the host range of the BRS virus compared to the HRS virus, and the role of the individual viral antigens required to elicit a protective immune response in the native host (currently HR
Provide an opportunity to study S virus). 2.2. F protein maturation BRS virus F protein consists of two polypeptide F ₁ and F ₂ linked by a disulfide bridge (Lerch
et al., 1989, J. Virol. 63: 833-840). There are differences in the mobility of SDS-polyacrylamide gel electrophoresis of F protein of BRS virus and HRS virus (Lerc
h et al., 1989, supra). Polyclonal and most monoclonal antibodies react with the F protein of both HRS and BRS viruses (Stott et al., 19
84; Orvell et al., 1987; Kennedy et al., 1988, J.
Gen. Virol. 69: 3023-3032; Lerch et al., 1989, supra). Paramyxovirus fusion protein F causes fusion of the virus to cells and fusion of infected cells to surrounding cells. Structurally, the F proteins of the various paramyxoviruses are similar to each other. F protein is synthesized as a precursor F _0, which is cleaved by hydrolysis with an internal hydrophobic region to produce two polypeptides F ₁ and F _2, it is disulfide bonds to form the active fusion protein. Carboxy-terminal hydrophobic region of F ₁ polypeptide, the amino terminus and outside the carboxy terminus inside the cell, is believed to fix the F protein membrane. The F protein is N-glycosylated (Mo
rrison, 1988, Virus Research 10: 113-136).
The HRS virus F protein is a typical paramyxovirus fusion protein. Antibodies specific for the F protein block fusion of infected cells (Walsh and Hruska, 19
83, J. Virol. 47: 171-177; Wertz et al., 1987, J. Vi.
rol. 61: 293-301), but may further neutralize the infectivity of the virus (Fernie and Gerin, 1982, Inf. Immun. 37: 243-
249; Walsh and Hruska, 1983, J. Virol. 47: 171-177;
Wertz et al., 1987, J. Virol. 61: 293-301), does not inhibit adhesion (Levine et al., 1987, J. Gen. Virol. 68:
2521-2524). F protein is synthesized as a polypeptide precursor F _0, which is cut into two polypeptides F ₁ and F _2. These two polypeptides are disulfide-linked and N-glycosylated (Fernie and Ger.
in, 1982, Inf. Immun. 37: 243-249; Lambert and Levi.
ne, 1983, J. Gen. Virol. 64: 825-832; Lambert and P
ons, 1983, Virol. 130: 204-214). 2.3. Bovine RS virus vaccine Bovine RS virus (BRS) vaccines have been developed that contain live or inactivated viruses, or viral proteins. Frennet et al. (1984, A
nn. Med. Vet. 128: 375-383) showed that 81% of calves given a mixture of live BRS virus and bovine virus diarrhea vaccine were protected from severe respiratory symptoms induced by field virus challenge. Reported. Stott et al. (1984,
J. Hyg. 93: 251-262) is an inactivated BRS virus vaccine (containing glutaraldehyde-fixed bovine nasal mucosal cells permanently infected with BRS virus and emulsified with oil adjuvant) and two live vaccines ( One against BRS virus and the other against HRS virus). In Stott's experiment (supra), 11 of the 12 calves that received the inactivated virus vaccine had B
Although completely protected from respiratory syncytial virus challenge, all control animals and animals receiving the live vaccine were infected. Live vaccines can exacerbate BRS virus infection.
An outbreak of respiratory disease associated with the BRS virus occurred shortly after inoculation of calves with the modified live BRS virus (Kimman et al., 1989, Vet. Q. 11: 250-253). Pa
rk et al. (1989, Res.Rep.Rural Dev.Adm. 31: 24-29)
Is a binary ethyleneimine (BEI) -inactivated BRS
We reported the development of a viral vaccine and tested its immunogenicity in guinea pigs and goats. Serum neutralizing antibodies were detected 2 weeks after inoculation, and antibodies were increased by booster injection at 4 weeks. In goats, a protective effect against the BRS virus was observed when the animals were challenged with the virus 12 weeks after inoculation. Trudel et al. (1989, Vaccine 7: 12-16) reported that humans (Lon
g) The ability of the immunostimulatory complexes prepared by adsorbing surface proteins of both RS strains of bovine (A-51908) to the adjuvant Quil A to induce neutralizing antibodies was studied. Immunostimulatory complexes made from bovine RS virus proteins were found to be significantly more effective at inducing neutralizing antibodies than their human counterparts.

[Means for solving the problem] 3. Summary of the Invention The present invention relates to a recombinant encoding a BRS virus protein.
DNA molecules, BRS virus proteins and pepti
And recombinant BRS virus produced therefrom
It concerns vaccines. It is partially and completely
Substantially all encoding a novel BRS virus F protein
Based on cloning of long cDNAs. F cD
The nucleotide sequence of NA was determined and is shown in FIG.
According to a particular aspect of the present invention, the BRS virus protein
DNA encoding the protein or peptide is BRS virus
It can be used to diagnose infection and
(Vaccinia virus or bacteria, yeast, insects or
Including but not limited to other vertebrate vectors)
Can also be inserted. These expression vectors are BR
Produce large quantities of S virus proteins or peptides
And the substantially pure obtained in this way.
Viral proteins or peptides are subunits
In a chin formulation or for diagnosis of BRS virus infection
Monoclonal or polyclones that can be used for isolation and passive immunization.
Used to generate clonal antibodies. another
In an embodiment, the tans of the BRS virus obtained according to the invention is
The protein sequence can be subunit vaccine or monoclonal
Or synthetic peptides that can be used for the production of polyclonal antibodies
Used to produce tides or proteins. Ma
In addition, non-pathogenic expression vectors are themselves recombinant viruses.
Used as a vaccine. 4. Detailed description of the invention The present invention relates to bovine respiratory syncytial virus nucleic acids and proteins.
You. Clarify the disclosure rather than limit the invention
To that end, a detailed description of the present invention is provided in the following subsections.
Divided. (ii) Bovine RS virus gene cloning; (ii) Bovine RS virus protein expression; (iii) Genes and proteins of the invention; and (iv) Utility of the invention 4.1. Cloning of bovine respiratory syncytial virus gene According to the present invention, cDNA conversion of BRS virus mRNA
Clones the transcripts and clones the full-length BRS virus
By identifying the protein-coding sequence,
Alternatively, obtain from plasmid pRLF2012-76-192.
Using the probe shown or from the sequence shown in FIG.
Using designed oligonucleotide probes
To identify the nucleic acid sequence encoded by the BRS virus
Now encodes the BRS virus protein
Bovine respiratory syncytial virus inheritance, defined as a nucleic acid sequence
The offspring can be identified. For example, in limiting
BRS virus corresponding to a specific protein
Includes complete open reading frame for RNA
The cDNA is a BRS virus mRNA template and a second
Using specific synthetic oligonucleotides for strand synthesis
Synthesized. Oligonucleotide nucleotide sequence
Is determined by sequence analysis of BRS virus mRNA
You. First-strand synthesis was described by D'Alessio et al. (1987, Focus 9 : 1
Perform as described in -4). After synthesis, RNA
RNaseA (at least about 1 μg / μl
Digest with l). Subsequently, the resulting single-stranded cDN
A is isolated by phenol extraction and ethanol precipitation
You. Oligonucleotides used for second strand synthesis are related
Specific sequence at the 5 'end of the viral mRNA
Preferably, and to facilitate cloning.
Contains a restriction endonuclease cleavage site useful for
Is preferred. Subsequently, the single-stranded cDNA was
Mix with Reotide (about 50μg / ml) and heat at 100 ℃ for 1 minute
And put on ice. Then, using reverse transcriptase, 50 mM T
ris-HCl (pH 8.0), 50 mM KCl, 5 mM MgCl _Two , 10mM
Osleitol, 1.6 mM dNTPs and 100 U reverse transcriptase
For about 1 hour at 50 ° C in a reaction mixture consisting of
To synthesize the second strand of the cDNA. Then, phenol
The cDNA is separated from the protein by extraction and ethanol
Collected by precipitation and blunt-ended with T4 DNA polymerase.
To the end. Subsequently, the blunt-ended cDNA was digested with the appropriate restriction
Donuclease (e.g., oligonucleotide primers
Sequence that recognizes the cleavage site of
Is not recognized).
Protein and subsequently cloned into a suitable vector
Become In addition, cDNA generated from viral mRNA
Was cloned into a vector, and the FRS
Contains a part of the nucleotide sequence shown in FIG.
Using oligonucleotides as probes,
A clone carrying a nucleic acid sequence is created. Virus
The cDNA library is, for example, Grunstein and Hogness
(1975, Proc. Natl. Acad. Sci. USA)
Screened using the method described. Then recovered
Clones into restriction fragments well known in the art
• Mapping and sequencing methods (eg, Sanger et al., 197
9, Proc. Natl. Acad. Sci. USA 72 : 3918-3921)
Analyze more. Or, all or one of the desired genes
The part is chemically synthesized based on the sequence shown in FIG.
In yet another embodiment, the nucleic acid molecule of the invention and
And / or the nucleic acid sequence shown in FIG. 2, or the amino acid sequence shown in FIG.
A protein derived from a nucleic acid sequence that substantially encodes an acid sequence
Limer with polymerase chain reaction (P
CR); Saiki et al., 1985, Science 230 (: 1350-1354)
Used for BRS virus proteins or related
Generate a nucleic acid molecule that encodes the peptide. PCR
Requires a primer and sense strand. Obedience
Thus, one of the BRS virus nucleic acid or amino acid sequences
Degenerate oligonucleotide primers corresponding to segments
-With the primer of one DNA strand (for example, the sense strand)
BRS virus nucleic acid or amino acid sequence
Another degenerate orientation homologous to the second segment of the row
The oligonucleotide primer is attached to the second DNA strand (eg,
Used as primer for antisense strand). Preferably
Can be used to replace these primers with a contiguous portion of a known amino acid sequence.
Selection based on the number of
Related DNA reaction products resulting from the use of
The expected size (i.e., expressed in base pairs)
The product length is the length of the two primers and the length of the two primers.
Tongue sandwiched between segments corresponding to limers
3 times the number of amino acid residues in the protein segment
Total). Subsequently, the BRS virus nucleic acid sequence,
Or cDN prepared from BRS virus mRNA
Using primers in PCR with nucleic acid template consisting of A
You. The DNA reaction product can be prepared by any method known in the art.
Clone using Numerous known in the art
Vector-host systems can be used. It is possible
Vectors can be cosmids, plasmids or mutated
Including, but not limited to, Ruth. But vector
The system must be compatible with the host cell used.
Such vectors contain bacteriophage such as ram
Derivatives, or plasmids such as pBR322, pUC or
Contains Bluescript R (Stratagene) plasmid derivative
But not limited to them. The recombinant molecule is transformed,
Transfection, infection, electroporation, etc.
Into the host cell. BRS virus gene
Insert into the roning vector and use this vector
Transform or transfect appropriate host cells
Or infect, and as a result,
Many copies are produced. This is a DNA fragment
Into a cloning vector with complementary cohesive ends
Achieved by ligating. However,
Complementary regimes used to fragment DNA
If the restriction site is not present in the cloning vector,
The end of the DNA molecule is modified with an enzyme. Also, nucleo
Ligation of the peptide sequence (linker) to the DNA end
And can produce any desired site; these
Ligated linkers are restriction endonuclease certified
A specific, chemically synthesized orifice that encodes
Consisting of gonucleotides. In addition,
Primers must be suitable for restriction endonuclease cleavage
It can be genetically engineered to include the site. Otherwise
And truncated vector and BRS virus gene
May be modified by adding a homopolymer. Specific implementation
In an embodiment, the isolated BRS virus gene, cD
Recombinant DNA containing NA or synthetic DNA sequence
Transformation of the host cell with the offspring results in the production of high copy number genes.
Enable Therefore, transformants can be grown and transformed.
Isolate the recombinant DNA molecule from the recombinant and, if necessary,
Recovering the inserted gene from the isolated recombinant DNA
Thus, a large amount of gene can be obtained. 4.2. Expression of bovine RS virus protein Encoding a BRS virus protein or part thereof
Nucleotide sequence into a suitable expression vector (i.e.,
Required for transcription and translation of the protein-encoding insert
Vector containing the element). Necessary transcription and translation
The translation signal is also provided by the native BRS virus gene.
Be paid. Proteins using various host / vector systems
Is expressed. These are viruses
(E.g., vaccinia virus, adenovirus, etc.)
Stained mammalian cell lines; viruses (eg, baculo
Insect cells infected with ills; yeast containing yeast vectors
Microorganisms such as mother, or bacteriophage DNA,
Transformed with plasmid DNA or cosmid DNA
Including but not limited to bacteria. These
The expression elements of the vector differ in their strength and specificities.
Depending on the host / vector system used, a number of suitable
Any of the transcription and translation elements may be used. DN
Any of the above for inserting the A fragment into the vector
The appropriate transcription / translation control signal and tag
Includes chimeric genes consisting of protein-encoding sequences
Construct an expression vector. These methods are recombinant in vitro
DNA, synthetic technology and in vivo recombination (genetical recombination
E). BRS virus protein or pepti
Expression of the nucleic acid sequence encoding the fragment
Of the BRS virus.
Protein or peptide is transformed with the recombinant DNA molecule
Expressed in the transformed host. For example, BRS virus
Protein expression can be determined by any of the protocols known in the art.
It may be controlled by a motor / enhancer element. BR
Regulates S virus protein or peptide expression
The promoter used for this is the CMV promoter
(Stephens and Cockett, 1989, Nucl. Acids Res. 17 : 71
10), SV40 early promoter region (Bernoist and Chambo
n, 1981, Nature 290 : 304-310), Rous sarcoma virus
Promo included in 3 'Long Terminal Repeat
(Yamamoto, et al., 1980, Cell twenty two : 787-797),
Herpes thymidine kinase promoter (Wagner
etal., 1981, Proc. Natl. Acad. Sci. USA 78 : 144-
1445), the regulatory sequence of the metallothionein gene (Brinster e
t al., 1982, Nature 296 : 39-42); Prokaryotic expression vector
Promoter, such as the β-lactamase promoter (Villa-
Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. US
A. 75 : 3727-3731), or tac Promoter (DeBoer, et
al., 1983, Proc. Natl. Acad. Sci. USA 80 : 21-2
5) Also Scientific American, 1980, 242 : 74-94; “Us
See also “eful proteins from recombinant bacteria”;
Nopaline synthase promoter region (Herrera-Estrella
et al., Nature 303 : 209-213) or Cauliflower Mo
Zyke virus 35S RNA promoter (Gardner, e
t al., Nucl. 1981, Acids Res. 9 : 2871), and optical coupling
Synthase, ribulose biphosphate carboxylar
Ze promoter (Herrera-Estrella et al., 1984, N
ature 310 : 115-120); Gal 4
Promoter, ADC (alcohol dehydrogener
Ze) promoter, PGK (phosphoglycerol / kina
) Promoter, alkaline phosphatase pro
Promoters from yeast or other fungi such as motors
Elements and tissue specificity and are transgenic
The following animal transcriptional regulatory regions used in the wild animal: pancreas
Elastase I gene regulatory region active in splanchnic acinar cells (S
wiftet al., 1984, Cell 38 : 639-646; Ornitz et al.,
1986, Cold Spring Harbor Symp, Quant. Biol. 50 : 399-4
09; MacDonald, 1987, Hepatology 7 : 425-515); pancreas
Insulin gene regulatory region (Hana
han, 1985, Nature 315 : 115-122), active in lymphoid cells
Grosschedlet a
l., 1984, Cell 38 : 647-658; Adames et al., 1985, Na
ture 318 : 533-538; Alexander et al., 1987, Mol. Cel
l. Biol 7 : 1436-1444), testis, breast, lymphoid cells and
Mouse mammary tumor virus regulatory region active in mast cells
(Lederet al., 1986, Cell 45 : 485-495), active in liver
Regulatory region of albumin gene (Pinkert et al., 1987,
Genes and Devel. 1 : 268-276), α-fe
Topoprotein gene regulatory region (Krumlarf et al., 1985,
Mol. Cell. Biol. 5 : 1639-1648; Hammer et al., 198
7, Science 235 : 53-58); α-1-anti active in liver
Trypsin gene regulatory region (Kelsey et al., 1987, Gen.
es and Devel. 1 : 161-171), β-g active in bone marrow cells
Robin gene regulatory region (Mogram et al., 1985, Nature 315 :
338-340; Kollias et al., 1986, Cell 46 : 89-94), brain
Myelin basic protein is active in oligodendrocytes in rats
Gene regulatory regions (Readhead et al., 1987, Cell 48 : 703-7
12); Myosin L chain 2 gene regulatory region active in skeletal muscle
(Sani, 1985, Nature 314 : 283-286), active in the hypothalamus
Gonadotropin-releasing hormone gene regulatory region (Mason et al.,
1986, Science 234 : 1372-1378)
Not determined. Expression vector containing BRS virus gene insert
Are identified by three general approaches:
(a) DNA-DNA hybridization, (b) "marker"
Presence or absence of gene function, and (c) insertion sequence
Expression. In the first approach, the insertion into the expression vector is performed.
The presence of the inserted foreign gene indicates that the inserted BRS virus remains.
DNA-DN using a probe containing a sequence homologous to a gene
Detected by A hybridization. The second approach
In this case, the insertion of a foreign gene into a vector
Specific “marker” gene function (eg, thymidine
・ Kinase activity, antibiotic resistance, transformation expression
Type, inclusion body formation in baculovirus, etc.) or
Identify and select recombinant vector / host systems based on absence
I do. For example, the BRS gene can be
A recombinant containing a BRS insert when inserted into a child sequence
Are identified by the absence of marker gene function. Third
In the approach described above, foreign genes expressed by recombinant
Recombinant expression vector by assaying offspring products
Is identified. Identify and isolate specific recombinant DNA molecules
Et al., Using several methods known in the art.
To grow. Once a suitable host system and growth conditions have been established,
The recombinant expression vector is propagated and produced in large quantities.
As described above, the expression vector used is the following vector
Or their derivatives, including but not limited to
I: To give a few examples, vaccinia virus or
Human or animal viruses such as adenovirus, especially
Bovine adenovirus and bovine herpes virus;
Insect virus such as baculovirus; yeast vector
ー; bacteriophage vectors (eg lambda) and
And cosmid DNA vectors. Further
In addition, adjust the expression of the inserted sequence or
A host that modifies and processes gene products in a defined manner
A cell line may be selected. Departure from a specific promoter
Reality is enhanced in the presence of certain inducers;
Controlling expression of gene-engineered BRS virus proteins
I can control. In addition, individual host cells are responsible for protein translation.
And post-translational processing and modification (e.g., glycosylation
Has a unique and unique mechanism for
are doing. For example, expression in bacterial systems is
Used to produce the oxidized core protein product
You. Expression in mammalian cells is a heterologous BRS virus protein.
Ensuring "natural" glycosylation of protein
Used for In addition, different vector / host expression systems
Varies processing reactions such as proteolytic cleavage
It can be done to any degree. Releases BRS virus gene
Once the emerging recombinants have been identified, the gene products are analyzed.
Should. This is the physical or functional nature of the protein
Achieved by a property-based assay. BRS ui
Once a virus protein or peptide has been identified,
Matography (e.g., ion exchange, affinity
-, Size column chromatography), centrifugation, fractionation
By standard methods including solution or for protein purification
It is isolated and purified by any of the other standard methods.
In yet another embodiment of the present invention, a BRS virus
A protein or peptide that is well known in the art
It is manufactured by chemical synthesis using the method described above. Such person
The law is described, for example, in Barany and Merrifield (1980, in “The
Peptides, "Vol. II, Gross and Meienhofer, eds.,
J. Academic Press, NY pp. 1-284).
You. Furthermore, recombinant but non-pathogenic viruses that infect cattle
Virus (vaccinia virus, bovine herpes virus and
And bovine adenovirus, including but not limited to
Gene) and use suitable promoter elements to
Express the BRS virus protein. Such pairs
The recombinant virus is used to infect cattle, thereby
Immunity to the S virus can be achieved. Further
Can express the BRS virus protein,
A recombinant virus as a component of a vaccine
It can also be deactivated before doing so. 4.3. Genes and proteins of the invention In the method described above and in the examples section 6 and 7 below
Using the method described, the following nucleic acid sequence was determined and
The amino acid sequences corresponding to them were deduced. BRS Will
The cDNA sequence of the protein is determined and the corresponding m
The RNA sequence is shown in FIG. These arrays or with them
Each functionally equivalent is used in accordance with the present invention.
The present invention further provides a B comprising at least 10 nucleotides.
Sequence of nucleic acid encoding RS virus F protein and
And subsequences are, for example, nucleic acid hybrids.
Assay, Southern and Northern blot analysis, etc.
Nucleic acid sequence encoding BRS virus F used for
And a portion capable of forming a hybrid. The present invention also
Amino acid sequence described in 3 or functional with them
Matches the equivalent of or has been deposited with the ATCC
encoded by cDNA clone pRLF2012-76-192
BRS virus F protein, its fragment
And derivatives. The invention also includes antigenic determinants.
Fragment or induction of BRS virus F protein
Provide conductors. For example, the nucleic acid sequence shown in FIG.
Sequences may contain substitutions, additions or deletions that result in functionally equivalent molecules.
Can be changed by loss. Nucleotide code
Due to the degeneracy of the sequence, the amino acid sequence shown in FIG.
Other DNA sequences encoding substantially the same sequence are also contemplated by the present invention.
It is used to carry out. These are the BRs described in FIG.
Nucleotide sequence encoding S virus F protein
But functions within the sequence to produce silent mutations
Of different codons that code for equivalent amino acid residues
All or part of the nucleotide sequence altered by the exchange
Including, but not limited to, a nucleotide sequence consisting of
No. Similarly, the BRS virus F protein of the present invention or
Is the fragment or derivative of the primary amino acid sequence
Substantially all of the amino acid sequence described in FIG.
Or containing a part or producing a silent mutation
Residues in the sequence with functionally equivalent amino acid residues
Coded by pRLF2012-76-192 containing the changed sequence
Including, but not limited to, example
For example, one or more amino acid residues in a sequence may be referred to as a functional equivalent.
Other sources of similar polarity that act
Replace with amino acids. Amino acid substitutions within the sequence
It is selected from other members of the class to which the amino acid belongs.
For example, non-polar (hydrophobic) amino acids are alanine and leucine.
, Isoleucine, valine, proline, phenylalanine
, Tryptophan and methionine. Polar neutral
Amino acids are glycine, serine, threonine and cysteine
And tyrosine, asparagine and glutamine.
Positively charged (basic) amino acids are arginine and lysine.
And histidine. Negatively charged (acidic)
Minic acids include aspartic acid and glutamic acid. Transliteration
During or after translation, eg, glycosylation, protein
Degradation cleavage, binding to antibody molecules or other cellular ligands, etc.
Virus proteins differentially modified by
Or fragments or derivatives thereof are also within the scope of the present invention.
Included in the box. In addition, BRS virus proteins
Recombination of the invention to alter processing or expression
The nucleic acid sequence encoding the BRS virus F protein
Genetic manipulation is also possible. For example, to limit
There is no sequence encoding the BRS virus F protein.
A signal sequence was inserted upstream of the sequence, and BRS virus F
Secretes protein, which can be harvested or biologically
Facilitates availability. Furthermore, facilitates another in vitro modification
To obtain the desired BRS virus protein
The nucleic acid sequence to be mutated in vitro or in vivo.
To create a translation initiation and / or termination sequence and
Destroys or mutates the coding region
And / or new restriction endonuclease sites
Can produce or destroy existing ones
You. In vitro site-directed mutagenesis (Hutchinson, et.
al., 1978, J. Biol. Chem. 253: 6551), TAB R linkers
(Pharmacia), including but not limited to
What kind of mutagenesis is known in the art
Can also be used in the law. 4.4. Utility of the invention 4.4.1. Bovine RS virus vaccine The present invention provides a safe and effective BRS virus vaccine.
Available for manufacturing. According to the present invention,
The term chin refers to an immune response induced against components of a preparation
Is understood to refer to a preparation that causes Advantages of the present invention
Are used in vaccines or passive immunization protocols
BRS virus to produce antibodies used in
The ability to produce large quantities of protein and immunogenic B
Non-pathogenic recombination producing RS virus protein
To provide a virus vaccine. these
Bovine disease previously exposed to the BRS virus by selective techniques
Modified BRS virus that may worsen the condition
The use of live vaccines can be avoided. According to the present invention,
BRS virus protein is produced in large quantities
The BRS virus nucleic acid sequence is inserted into a suitable expression system.
In certain embodiments of the invention, the BRS virus F
Protein is useful for subunit vaccines.
A large amount can be produced by the method described above. Specific preferred embodiments of the present invention
In an embodiment, the BRS virus F protein is rVVF4
64 (F protein producing virus)
Expressed by unrecombinant recombinant vaccinia virus
Harvested and subsequently in a preferred pharmaceutical carrier
It is administered as buunit. Or vaccinia wi
Rus, bovine herpes virus and bovine adenowis
BRS virus non-pathogenic to bovine and bovine
Including but not limited to retroviruses that express proteins
Infect cattle with an undefined recombinant virus.
Immunity to BRS virus without associated disease
Raise the epidemic. In yet another embodiment of the present invention,
Respiratory syncytial virus protein or peptide
Can be chemically synthesized for use. BRS Will
Vaccine containing peptide fragments of protein
In, select a peptide that is likely to cause an immune response
It is desirable. For example, BRS virus protein
Computer analysis of the amino acid sequence of
Without limitation, anti-hepatitis B virus surface antigen
Hopp and Woo have been successfully used to identify protogenic peptides
ds (1981, Proc. Natl. Acad. Sci. USA 78 : 3824-38
Using the method described in 28), the surface epitope is
Identify. To increase immunogenicity, e.g., pepti
Compound or conjugate the peptide to the carrier molecule.
Modify the BRS virus peptide
Is desirable. The vaccine of the present invention can be administered intranasally or
Intra, intramuscular, subcutaneous or intravenous and preferably intratracheally
Through various routes, including but not limited to
It is administered together with a suitable pharmaceutical carrier by exclusion. An example
For example, but not limited to, Freund (complete or
Perfect), inorganic gels like aluminum hydroxide, also
Is a surface active substance such as lysolecithin, Pluronic
Polyol, polyanion, peptide, oily emulsion, key
Whole limpet hemocyanin, dinitrophenol
Or Bacille calmette-Guerin (BCG) or Coryn
The invention with an adjuvant such as ebacterium parvum
It is desirable to administer the subunit Wachikun. Prevention
To achieve objective and / or long-lasting immunity
May require multiple inoculations. 4.4.2. Antibodies to bovine RS virus protein In another embodiment, the nucleic acids, proteins or
Uses peptides to bind to BRS virus proteins
Developing monoclonal or polyclonal antibodies
Wear. Monochrome against BRS virus protein
To prepare a null antibody, culture the continuous cell line
Any method that provides for the production of a molecule may be used. example
For example, Kohler and Milstein (1975, Narure 256 : 495-497)
Can use the hybridoma method originally developed by
You. Antibody to BRS virus protein
Loans can be prepared by known methods. Recombination
DNA method (for example, Maniatis et al., 1982, Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York)
Using a monoclonal antibody molecule or its antigen binding
A nucleic acid sequence encoding the region is constructed. Antibody molecule is known
Methods, such as immunoadsorption, immunoaffinity
Matography, HPLC (High Performance Liquid Chromatography
-) Or a combination thereof
The product is purified by straining. Antibodies containing the idiotype of the molecule
Fragments can be made by known methods. For example,
Such fragments can be used for pepsin digestion of antibody molecules.
F (ab ') generated from _Two Fragment; F (ab ') _Two flag
Produced by reducing the disulfide bridge of
Fab 'fragments and antibody molecules with papain
Fabs or Fas produced by treatment with an agent
b including but not limited to fragments. 4.4.3. Application to diagnosis Nucleic acids encoding BRS virus proteins
Proteins, peptide fragments or produced from them
Derivatives and BRS virus proteins and pep
The present invention relates to antibodies against tides.
Used to diagnose For example, the nucleic acid component of the present invention
Using the offspring, Northern blot analysis (in this analysis, dynamic
Hybridization of nucleic acid preparations obtained from products
Exposure to the complementary nucleic acid molecule of the invention under conditions
To detect hybridization)).
Presence of BRS virus nucleic acid in BRS virus infected animals
Is detected. For example, but not limited to, all RNs
A was obtained from a cow potentially infected with the BRS virus.
Nasal tissue swab, tracheal swab or optional top
It can be prepared from isolates from airway mucosal surfaces. continue,
Northern blot analysis of RNA (BRS virus nucleic acid
Obtained (e.g., radiolabeled)
Hybridized oligonucleotide probe
Northern blots carrying bovine RNA under mild conditions
Luther; after hybridization, filter
Wash and autoradiate probe binding to filter
(Visualized by chromatography). Or diagnostics
Present if the level of BRS virus in the sample is low
In order to amplify the amount of BRS virus nucleic acid, the present invention
BRS virus nuclei using gonucleotides in PCR reactions
It is desirable to detect the presence of the acid. Preferred of the present invention
In an embodiment, the viral RNA amplified from the cultured cells
By dot blot hybridization to RS virus
Analyze for sequence. The product of such a PCR reaction
BRS virus sequence not supplied by primers in
May be indicative of a BRS virus infection. another
In an embodiment, the BRS virus protein of the invention
And peptides are used to diagnose BRS virus infection
You. For example, but not limited to, the BRS virus
Proteins and peptides are available from anti-BRS virus antibodies.
Enzyme-linked immunosorbent assay (ELIS) to detect the presence
A), immunoprecipitation, rosette or Western blot
Used in the law. In a preferred embodiment, the anti-BRS
Viral antibody titer rises with active BRS virus infection
It shows. According to these embodiments, the serum test
Allows binding of antibodies to proteins or peptides
BRS virus protein or pepti
Exposure to antibodies to bind to the protein or peptide.
Is detected. For example, but not limited to, BR
Immobilize S virus protein or peptide on solid surface
To allow binding of antibodies to proteins or peptides.
May contain anti-BRS virus antibody
Exposure to a serum (test serum) followed by antibody BRS
Detects binding to irs protein or peptide
Activating substances, such as detectably labeled anti-immunity
Expose to globulin antibody. Or BRS virus
Analyze proteins or peptides for Western blot analysis
The western blot is then exposed to the test serum and
Body binding to BRS virus proteins or peptides
Is detected as described above. Yet another non-limiting of the present invention
In a specific embodiment, the BRS virus protein or
Or peptides are adsorbed on the surface of red blood cells,
Antigen-coated red blood cells may contain anti-BRS virus antibodies
Expose to certain serum. Rosette of antigen-coated erythrocytes with serum
Formation is due to exposure to BRS virus or active
It indicates that the infection was caused. Another embodiment of the present invention
In a similar manner, it indicates active BRS virus infection
Yes, detect the presence of BRS virus protein
Therefore, antibodies that recognize the BRS virus protein,
Use for ELISA or Western blot.

[Embodiment] 5. Example : Analysis of mRNA of bovine RS virus fusion protein and nucleotide sequence of recombinant vaccinia virus 5.1 Materials and methods 5.1.1 Virus and cells BRS virus 391-2, wild-type (Copenhagen strain) and recombinant vaccinia virus, The growth and proliferation of bovine turbinate (BT) cells, HEp-2 cells, and thymidine kinase-negative (tk-) 143B cells were determined by Hr
uby and Ball (1981, J. Virol. 40: 456-464; Stott et.
al., 1986, J. Virol. 60: 607-613; Lerch et al., 1989,
J. Virol. 63: 833-840). 5.1.2 Protein labeling of BRS virus infected cells
And harvest bovine nasal turbinate (BT) cell monolayers were simulated with BRS
Infected with virus or HR virus. Tunicamycin (1.5 μg / ml, Boehringer Mannheim) was added to the medium covering the cells 25 hours after infection, if necessary. One hour later, the medium was removed from all monolayers and replaced with methionine-free DMEM (Gibco). Tunicamycin was re-added if needed. After a 30 minute incubation,
[ ³⁵ S] methionine (100 μCi / ml, New England Nuclear) was added to the medium. 2 cells
Incubate for hours and allow the protein to
989, J. Virol. 63: 833-840. Virus-specific proteins were analyzed by Wertz et al. (1985, Proc. Natl. Acad. Sci. USA 82: 4075-407).
As described in 9), Welcome's anti-
Immunoprecipitation was performed using RS serum (Welcome Reagents). The protein was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 197
0, Nature (London) 227: 680-685) and a fluorometric method (Davis and Wertz, 1982, J. Virol. 41: 821-83).
Detected by 2). 5.1.3 cDNA synthesis, molecular cloning, and F
Identification of heterologous cDNA clones Using the strand replacement method of Gulber and Hoffman (1983, gene 25: 263-260), D'Alessio et al (1987, Focu
s 9: 1-4) cDNA was synthesized as described in The ends of the cDNA were blunted using T4 DNA polymerase (BRL) (Maniatis et al.,
1982, in "Molecular Cloning: a Laboratory manual," Co
ld Spring Harbor Laboratory, Cold Spring Harbor, N.
Y.). cDNA is M13mp19 replicative (RF) DN digested with SmaI and treated with bovine intestinal alkaline phosphatase (Maniatis et al., supra).
A. and competent E. coli. ColiDH5α
F 'cells (Bethesda Research Laboratory) were transfected (Hanahan, 1983, J. Mol. Bio
l. 166: 557-580). M13mp19 phage containing the BRS virus F-specific insert was nick-translated (Rigby et al., 1977, J. Mol. Biol. 113: 237-).
251), a previously identified BRS virus F gene-specific clone (Lerch et al., 1989, J. Vir.
ol. 63: 833-840) as a probe for dot blot hybridization of phage DNA (Davis et al., 198).
6, "Basic Methods in Molecular Biology. Elsevier Sc
ience Publishing Co., Inc., Ney York, NY). Propagation and manipulation of M13mp19 and recombinant phage were performed as described by Messing (1983, Meth. Enzymol. 101: 20-78). 5.1.4 Nucleotide sequencing and RNA
The dideoxynucleotide sequencing using the Klenow fragment of DNA polymerase I (Pharmacia) or modified T7 DNA polymerase (Sequenase, U.S. Biochemicals) of immersed E. coli was performed by Tabor and Rib.
chardson (1987, Proc. Natl. Acad. Sci. USA 84:47
46-4771) and Lim and Pene (1988, Gene Anal. T
ech. 5: 32-39)
M13 sequencing primers (New England Biolabs) were used. BRS virus F
Extension of a synthetic DNA primer that is complementary to bases 264 to 284 of the mRNA results in avian myeloblastosis virus (AM)
V) using reverse transcriptase (Molecular Genetic Lysis) (Air, 1979, Virology 97: 468).
-472). BRS virus mRNA used as a template in primer extension on RNA was harvested as described for mRNA used for cDNA synthesis (Lerch et al., 1989, J. Virol. 63: 833). -840
). Nucleotide sequencing and primer extension
Using [α- ³⁵ S] dATP (Amersham) and polyacrylamide-urea gradient gel electrophoresis, Bi
ggin et al. (1983, Proc. Natl. Acad. Sci. USA 8
0: 3963-3965). Nucleotide sequences were analyzed using software products from the Genetic Computer Group at the University of Wisconsin (Devereux et al., 1984, Nucl. AcidsRes.
s. 12: 387-395). 5.1.5 Complete c for BRS virus F mRNA
DNA Synthesis and Cloning cDNA containing the complete major open reading frame of the BRS virus F mRNA was synthesized using specific synthetic oligonucleotides for second strand synthesis. The nucleotide sequence for this oligonucleotide was determined from sequence analysis of BRS virus mRNA. First strand synthesis
As described in D'Alessio et al. (1987, Focus 9: 1-4). Following synthesis, the RNA template was digested with RNase A (1 μg / μl) at 37 ° C. for 30 minutes. The resulting single-stranded cDNA was isolated by phenol extraction and ethanol precipitation. Second
Oligonucleotides used for strand synthesis have the sequence 5 ′
CACGGATCCACAAGTATGTCCAACC
It has a 3 'and the first 9 bases 5' of the oligonucleotide contain a BamHI restriction site. Single-stranded c
DNA was mixed with this oligonucleotide (50 μg / ml), heated to 100 ° C. for 1 minute and placed on ice. AM
Using reverse transcriptase of V, reactants [50 mM Tris-HCl (pH 8.0), 50 mM KCl, 5 mM MgCl ₂ , 10 mM dithiothreitol, 1.6 m
M dNTPs, 100 U AMV reverse transcriptase] to synthesize second strand cDNA, and the reaction was incubated at 50 ° C. for 1 hour. The cDNA was separated from the protein by phenol extraction, recovered by ethanol precipitation and blunt-ended with T4 DNA polymerase. The blunt-ended cDNA was then converted to the restriction enzyme BamHI (Bethesda Research Laboratories).
And further separated from the protein by phenol extraction. M13mp18 RF DNA was transferred to BamH
After digestion with I and SmaI and treatment with bovine intestinal phosphatase, this was added to the above cDNA, and further recovered by ethanol precipitation. This cDNA was ligated to a vector, and further, Hanahan (1983, J. Mol. Biol. 16
6: 557-580) were transfected into sensitive DH5αF 'cells (Bethesda Research Laboratories). 5.1.6 Preparation and production of recombinant vaccinia virus vectors
CDN corresponding to F mRNA of BRS virus and isolated BRS virus
A clone F20 was digested with BamHI and KpnI, T4DN to create blunt ends of the cDNA.
Treatment with A polymerase and pIBI76-192
Was subcloned into recombination plasmid pIBI76-192 by ligation into the unique SmaI site. This plasmid, pIBI76-192, was constructed according to Ball et al. (1986, Proc. Natl. Acad. Sci. USA 83: 246-250).
Similar to recombination plasmids as described in In the case of pIBI76-192, base pairs (bp) 1 to 1710 of the HindIII J fragment of vaccinia virus were inserted between the HindIII and SmaI sites of pIBI76 (International Technology). 280 bp, that is,
DNA containing the 7.5k promoter of vaccinia virus
Fragment from the vaccinia virus thymidine kinase (t
k) Gene (bp 502 to 1070 of HindIII J fragment, Weir and Moss, 1983, J. Virol. 46: 530-5)
37) EcoRI site (bp of HindIII J fragment)
670). The direction of this promoter is
The reverse of the transcription direction of the vaccinia virus tk gene,
It was like directing right-to-left transcription on a regular vaccinia virus map. The unique Smal site in pIBI76-192 is downstream of the major transcription start site of the 7.5k promoter. BRS virus F
Isolation of recombinant vaccinia viruses containing the mRNA sequence was performed using Lavi and Ektin (1981, Carcin
ogenesis 2: 417-423), except by Stott et al. (1986, J. Virol. 60: 607-61).
Performed as described in 3). 5.1.7 Characterization of Recombinant Vaccinia Virus Vector Vaccinia virus core DNA isolated from wild-type and recombinant viruses was purified from Esposito et al. (1981, J. Virol.
Meth. 2: 175-179) and prepared for Southern blot analysis. DN by restriction enzyme digestion, Southern blot analysis, and nick translation
Radioactive labeling of A was performed as described in Section 6 above. Metabolic labeling of proteins from cells infected with wild-type and recombinant vaccinia virus
The procedure was as above, except that the infected cells were exposed to the label for 3 hours starting from 3 hours post-infection. Proteins were analyzed by SDS-polyacrylamide gel electrophoresis using standard methods. Immunoprecipitation of virus-specific proteins has been described by Wertz et al.
985, Proc. Natl. Acad. Sci. USA 82: 4075-4079)
As described in the Welcome Anti-R
This was performed using S serum (Welcome Regents). For immunofluorescence, HEp-2 cells were grown on cover slips. Cells are infected with wild-type or recombinant vaccinia viruses (moi = 10) and 24 hours after infection, the cells are indirectly fluoresced by anti-BRS virus 391-2 serum as primary antibody. (Lerch et al., 1989, J. Virol. 63: 833-840), followed by fluorescently conjugated anti-bovine IgG (H +
L). Fluorescence was observed using a Nikon fluorescence microscope. 5.2 Results 5.2.1 Nucleic acid sequence and its comparison In order to determine the complete nucleotide sequence of the BRS virus F mRNA, the cD
The NA clones were isolated and their nucleotide sequences were further analyzed. The BRS virus F mRNA sequence was determined from eight clones independently derived from four separate cDNA synthesis reactions. The regions of the BRS virus F mRNA determined from the different clones are shown in FIG. Most of this sequence consists of three clones, FB
3, FB5 and F20. Clone F2
0 corresponds to the full length of the BRS virus F cDNA,
It was synthesized using an oligonucleotide specific for the 5 'end of BRS virus F mRNA. Inserts from clones FB3, FB5, and F20 were excised using the restriction enzymes PstI and KpnI that do not cut within the insert, subcloned into M13mp18 RF DNA, and the other end of the cDNA was excised. The sequence was determined. Furthermore, the restriction fragments of the inserted fragments were
Subcloning into RF DNAs 13mp18 and mp19 allowed the determination of the sequence of the middle part of the cDNA. Clone F20 was replaced with a restriction enzyme, AlwN
I, Pf1MI, EcoRV or HpaI, digested clones FB3 and FB5 with restriction enzymes EcoRI
Digested. 5 'of F mRNA sequence of BRS virus
Ends show mRN from cells infected with BRS virus
Determined by extension of the DNA oligonucleotide on A. The sequence for the oligonucleotide complementary to bases 267 to 284 of the BRS virus F mRNA was determined from the sequence provided by the cDNA clones. Clones F29, FB2, F138, and
FB1s are all 750 nucleotides or less and have not been extensively sequenced. The BRS virus F mRNA contained 1899 nucleotides excluding the polyadenyl tail (FIG. 2). The seven bases at the exact 5 'end of the mRNA could not be determined due to the strong stop signal in all four nucleotide reactions for each base during primer extension for the mRNA. BR
The sequence at the 3 'end of the S mRNA F mRNA is all H
Respiratory syncytial virus gene (Collins et al., 1986, Proc.
End of tl. Acad. Sci. USA 83: 4594-4598) and B
One of the two consensus gene end sequences found at the 3 'end of the GmRAN of the RS virus, I found out. FmRA of BRS virus
N has a single major open reading frame, which starts at the start codon starting at nucleotide 14 and extends to the stop codon at nucleotide 1736. Just before the 3 'polyadenylation region was a non-coding region of 161 nucleotides at the 3' end (Figure 2).
Nucleotide sequence of the BRS virus F mRNA
HRS virus A2 (Collins et al., 1986, Proc. Nat.
l. Acad. Sci. USA 83: 4594-4598) FmRNA,
Long (Lopez et al., 1988, Virus Res. 10: 249-26).
2), RSS-2 (Baybutt and Pringle, 1987, J. Gen.
Vir. 68: 2789-2796) and 18537 (Johnson an
d Collins, 1988, J. Gen. Virol. 69: 2623-628), compared to the published sequence for various F mRNAs.
The degree of nucleic acid homology between the sequences was determined (Table 1).

[Table 1] HRS viruses A2, Long and PSS-2 are subgroup A viruses, and 18537 is a subgroup B virus (Anderson et al., 19).
85, J. Infect. Dis. 151: 626-633; Mufson et al., 198.
5, J. Gen. Virol. 66: 2111-2124). The level of nucleic acid homology (71.5%) between the B mRNA and FRS virus F mRNAs is similar to the level of homology observed when comparing the F mRNAs of the two HRS virus subgroups (79%). I was BRS virus and HRS
The level of homology between both the F mRNAs of the virus and between the F mRNAs of the two HRS virus subgroups is the level of homology between the F mRNAs of the HRS viruses within the same subgroup (97 -98%). The level of nucleotide sequence homology between the BRS virus and the HRS viruses in the 3 'non-coding region of the F sequence was 37.5% as compared to 74.5% in the coding region of the F sequence. This is the level of homology in the 3 'non-coding and coding regions, which is 47% and 82%, respectively, when comparing the F sequences of the HRS viruses of subgroup A with the F sequences of the HRS viruses of subgroup B. Was similar to Mutants were present at nucleotides on two sites in the FmRNA sequence. In one clone, clone F20, nucleotides 455 and 473 were G and C, respectively, rather than A and G as observed in other clones and shown in the sequence diagram (FIG. 2). 5.2.2 Predicted amino acids of BRS virus F protein
Sequence and Comparison with the HRS Virus F Protein Sequence The reading frame of the BRS virus F mRNA
An amino acid polypeptide was predicted. The amino acid sequence of this polypeptide is shown below the mRNA sequence in FIG. The predicted molecular weight of the predicted BRS virus F polypeptide was 63.8 kDa. The predicted polypeptide hydropathy is:
The amino terminus and residues 5 corresponding to residues 1 to 26
A strong hydrophobic region near the carboxy terminus corresponding to 22 to 549 was shown (FIG. 3). The hydropathy curve and amino acid sequence show that the regions (domains) in the BRS virus F protein are similar to those described for the HRS virus F protein, the amino-terminal signal peptide region (residues 1-26), A carboxy-terminal anchor region (residues 522-549) and F1
And a putative cleavage sequence (residues 131-136) producing the F2 polypeptide (see FIGS. 3 and 4). There were three possible sites for the addition of N-linked hydrocarbon side chains, two of which were in the proposed F2 polypeptide and one in the proposed F1 polypeptide ( (Fig. 2). The predicted FRS amino acid sequence of the BRS virus was compared to HRS viruses, A2 (Collins et al., 1984, Proc. Natl. Acad.
Sci., USA 81: 7i683-7i687), Long (Lopez et.
al., 1988, Virus Res. 10: 249-262), RSS-2 (B
aybutt and Pringle, 1987, J. Gen. Virol. 68: 2789-
2796), and 18537 (Johnson and Collins, 198
8, J. Gen. Virol. 69: 2623-628) compared to the predicted amino acid sequences of the F polypeptides (FIG. 4). B
The F polypeptide of the respiratory syncytial virus has all four types of HRS
Like the virus F polypeptides, it is 574 amino acids. The proposed carboxy-terminal hydrophobic anchor (residue 522) present in the F protein of the BRS virus
-549) and the amino-terminal signal region (residues 1-2)
6) was similar to that present in the FRS proteins of the HRS virus. In addition, the Lys-Lys at residues 131 to 136 in the BRS virus F protein.
The sequence -Arg-Lys-Arg-Arg showed the proposed cleavage signal. This sequence was homologous to the proposed cleavage signals in all HRS virus F proteins. The cleavage sequence in the F protein of the BRS virus follows the stretch of hydrophobic residues (residues 137-158), which means the amino terminus of the F1 polypeptide after cleavage. Seem. Clone F
The nucleotide difference in 20 results in amino acid residue 1 being part of the proposed F1 amino terminus.
48 and 154, which are respectively Va
It changes from 1 to Ile and from Leu to Val. These differences result in the HRS virus F
Homologous to a correlated amino acid site in proteins,
It will occur in amino acids at these two sites in the F protein of the BRS virus. With the exception of one cysteine residue (residue 25) in the proposed amino-terminal signal peptide, all cysteine residues were conserved at sites within the F protein of BRS and HRS viruses. It contains a cysteine residue at position 550, which has been shown to be the site of covalent attachment of palmitic acid to the human RS virus F protein (Ar
umugham et al., 1989, J. Biol. Chem. 264: 10339-1034.
2). In the F1 polypeptide of the BRS virus F protein, there is a single possible site for N-linked glycosylation (residue 500), which is conserved in all HRS virus F proteins. (FIG. 4). In the BRS virus F2 polypeptide, N-linked glycosylation (residues 27 and 120)
There were two possible sites for (Figures 2 and 4). This possible site at residue 27 was conserved in the BRS and HRS virus F proteins (FIG. 4). However, the HRS virus F ₂
Polypeptides contain a total of four or five possible sites, the number of which depends on the isolate;
The position of the remaining potential site for N-linked glycosylation in the S virus F2 polypeptide was not conserved in all HRS virus F2 polypeptides (FIG. 4). On F ₂ polypeptides of BRS virus, the presence of only two possible N- linked glycosylation site is that differences in mobility on electrophoresis F ₂ polypeptides of BRS virus and HRS virus were present It was consistent with earlier observations (Lerch et al., 1989, J. Virol. 63: 833-840). Amino acid identity between F proteins of various viruses
Various regions of the F protein are shown (Table 2).

[Table 2] A2, Long and RS, which are HRS viruses
S-2 is a subgroup A virus,
8537 is a subgroup B virus (Anderson
et al., 1985, J. Infect. Dis. 151: 626-633); Mufso.
n et al., 1985, J. Gen. Virol. 66: 2111-2124). Although the largest mutated region is within the proposed signal peptide region (residues 1-26), the overall hydrophobicity of this region was conserved in the BRS virus F protein. (FIG. 3). Proposed F ₂ polypeptide of F protein of BRS virus, H
Compared to homology which exists between F ₂ polypeptides subgroups with RS virus A and B, showed a low level of homology than against F ₂ polypeptides (Johnso
n and Collins, 1988, J. Gen. Virol. 2623-2628). In contrast, the level of homology between the various F ₁ polypeptides can be compared BRS virus HRS virus, even try to compare the two HRS virus subgroup comrades cases Was similar. 5.2.3 Electrophoresis of BRS virus F protein
BRS virus F protein (Lerc) observed before the effect of tunicamycin on mobility
h et al., 1989, J. Virol. 63: 833-840) is an inhibitor of N-linked glycosylation to determine if it is really due to N-linked glycosylation. The electrophoretic mobility of the radioactively labeled BRS virus F protein in the presence and absence of tunicamycin was investigated. Proteins in BRS virus, HRS virus, and mock infected cells were isolated in the presence and absence of tunicamycin [ ³⁵
Radiolabeled by exposure to [S] methionine, immunoprecipitated, and further separated by SDS-polyacrylamide gel electrophoresis. The BRS virus F protein labeled in the presence of tunicamycin demonstrated a change in electrophoretic mobility compared to the labeled BRS virus F protein in the absence of tunicamycin (FIG. 5, lanes _BT and B). Further, F _0, F ₁ of the synthesized BRS virus in the presence of tunicamycin and, F ₂ polypeptide, F _0, F ₁ of HRS virus synthesized in the presence of tunicamycin, and, of F ₂ polypeptides Each had similar electrophoretic mobilities (FIG. 5, lanes H _T and B _T ). These results indicate that BR
The F protein of the S virus is glycosylated via N-linked carbohydrate addition, and
Differences observed in the electrophoretic mobility of F ₂ polypeptide with RS virus, as expected by the deduced amino acid sequence, there is shown that is due to differences in the extent of glycosylation (FIG. 4). 5.2.4 Recombinant vaccinia containing BRS virus gene
To facilitate the study of the role of BRS virus unique proteins in generating viral vectors and inducing a protective immune response in isolated hosts, the BRS virus F protein was placed in a vaccinia virus expression vector. . CDNA containing the complete major open reading frame of F mRNA of BRS virus
(F20) was replaced with pIBI76-1, a plasmid designed for the production of vaccinia virus recombinants
92. This plasmid pIBI76-1
92, Ball et al. (1986, Proc. Natl. Acad. Sci. U.
SA 83: 246-250), which resembles the thymidine kinase (t
k) Includes a portion of the Vaccinia virus HindIIIJ fragment with the 7.5K promoter inserted into the gene. However, in the case of pIBI76-192, this 7.5K promoter directs transcription in the opposite direction of transcription of the tk gene. The BRS virus FmRNA cDNA was inserted downstream of the major transcription start site of this 7.5K promoter. HindIII J containing the inserted BRS virus F gene
The fragment was homologously recombined (Stott et al., 1986, J. Virol.
60: 607-613) into the genome of the vaccinia virus (Copenhagen strain). Thymidine kinase negative recombinant vaccinia virus (rVV) was identified by hybridization of the recombinant virus using a probe specific for the F gene of the BRS virus and selected by three rounds of plaque purification. Recombinant vaccinia viruses F464 and F1597 each had 7.5
B in the forward and reverse directions with respect to the promoter of K
It contained the F gene of the RS virus. The genomic structure of the recombinant vaccinia virus was determined by Southern blot analysis of restriction enzyme digests of vaccinia virus core DNA. These experiments demonstrated that the F gene of the BRS virus was inserted into the tk gene of the recombinant viruses. 5.2.5 Recombinant waxes containing F gene of BRS virus
Protein from cells infected with near virus
The ability of recombinant vaccinia virus containing the FRS gene of the analyzed BRS virus to express the F protein of the BRS virus was examined in tissue culture cells. BT cells were infected with either the BRS virus, wild-type vaccinia virus, or recombinant vaccinia viruses containing the F gene of the BRS virus in a positive or negative orientation with respect to the promoter. The proteins in the cells were labeled by incorporation of [ ³⁵ S] methionine, collected, and then immunoprecipitated with the anti-RS serum from Welcome, and further purified by S
Separation was performed by DS-polyacrylamide gel electrophoresis. Recombinant vaccinia virus F464 (forward) produces at least two proteins in infected cells, and these proteins are transferred to Welcome Anti-R
S serum (FIG. 6, lane F46)
4+), these proteins are present in cells infected with wild-type vaccinia virus (FIG. 6, lane VV) or cells infected with rVVF1597 (reverse) (FIG. 6, lane F1597-). Did not exist. two proteins specific for a cell class infected with rVVF464 had a mobility on homologous electrophoresis F ₀ and F ₁ polypeptide of BRS virus. Cytoplasmic proteins precipitated by serum (FIG. 5, lane M)
Has a electrophoretic mobility similar to F ₂ polypeptide of the BRS virus and, rVV F464
It inhibited the detection of the F ₂ polypeptide in the infected allowed within cells. F ₀ protein is produced, in the case of producing a cleaved by F ₁ polypeptide, if not be visualized, it has been estimated that also present also F ₂ polypeptide. Previously observed in cells infected with the BRS virus (Lerch et al., 1989, J. Virol. 63: 833-840)
And an additional protein, slightly larger than the 22K protein of the BRS virus, was observed in cells infected with rVVF464 (FIG. 6, lane F46).
4+). This additional protein was produced only in cells infected with the BRS virus or cells infected with rVVF464, and was immunoprecipitated with the welcome antiserum. The results indicated that this additional protein could either be a specific cleavage fragment of the BRS virus F protein or interact with the BRS virus F protein. 5.2.6 BR expressed from recombinant vaccinia virus
Glycosylation of the S virus F protein Determines whether F polypeptides synthesized in cells infected with the recombinant virus are glycosylated in a manner similar to the true BRS virus F polypeptide. B infected with rVVF464 for purpose
Proteins in T cells were labeled with [ ³⁵ S] methionine in the presence and absence of tunicamycin.
These proteins were transformed with BRS virus-infected B
Similar labeled proteins from T cells were compared by immunoprecipitation and SDS-polyacrylamide gel electrophoresis (FIG. 7). In the presence of tunicamycin, the F ₀ and F ₁ polypeptides produced in cells infected with the rVV F464 virus have a faster electrophoresis compared to their counterparts synthesized in the absence of tunicamycin. It has mobility on electrophoresis (Fig. 7,
Compare lane F _T to lane F) and have electrophoretic mobilities homologous to unglycosylated F ₀ and F ₁ polypeptides from cells infected with BRS virus. (Figure 6, lanes F _T compared to lane B _T). In the presence of tunicamycin, the band of the protein from the cell such infected with recombinant virus, in addition to possibly lost cytoplasmic protein comprises F ₂ polypeptide of the BRS virus and, BRS virus non-glycosylated bottom of the gel where the F ₂ will migrate (Fig. 7, lane F _T) intensity of the bands was increased (see FIG. 5). 5.2.7 BR expressed from recombinant vaccinia virus
Cell surface expression of the F protein of the S virus The F glycoprotein of the HRS virus is expressed on the surface of infected cells and incorporated into the membrane of virions (Huang, 1983, "The genome and gene products of hu").
man respiratory syncytial virus "Univ. of North Ca
rolina at Chapel Hill; Huang et al., 1985, 2: 157-17
3). In order to determine whether the BRS virus F protein expressed in cells infected with the recombinant vaccinia virus is transported on the surface of the infected cells and expressed there, The allowed cells were examined by indirect immunofluorescence staining. BT cells were extremely sensitive to vaccinia virus infection and could not be used for immunofluorescence measurements without high background fluorescence. For this reason, immunofluorescence has
Performed on HEp-2 cells infected with the recombinant virus. The antiserum used in this immunofluorescence method was BRS
Is a virus 391-2 specific antiserum,
Recognition of the BRS virus F protein has previously been shown in Western blot analysis of proteins from cells infected with S virus (Lerch et al., 1989, J. Virol. 63: 833-). 840). This antiserum is specific for immunofluorescence assays on cells infected with the BRS virus, but shows no specificity for uninfected cells. HEp-2 cells infected with recombinant F-464 (FIG. 8, rVVF column) showed specific surface fluorescence, which was due to uninfected cells (FIG. 8, column M) or wild-type vaccinia virus. Were not present in any of the cells infected with (FIG. 8, column VV). 5.3 Conclusion We have determined that the cD corresponding to the BRS virus F mRNA
The nucleotide sequence of the NA clone was determined. HR correlating nucleotide sequence and deduced amino acid sequence
Compared to the S virus sequence. F mRNA is 1
At 899 nucleotides in length, it was homologous to the A2, Long and RSS-2 F mRNAs of the HRS virus (Collins et al., 1984, Proc. Natl. Aca.
d. Sci. USA 81: 7683-7687; Baybutt and Pringle, 1
987, J. Gen. Virol. 68: 2789-2796; Lopez et al. 198
8, Virus Res. 10: 249-262). HRS virus 1853
The 7 F mRNA is 3 nucleotides shorter in the 3 'non-coding region (Johnson and Collins, 1988, J. Mol.
Gen. Virol. 69: 2623-628). BRS virus and HR
The level of nucleic acid homology between the F mRNA with the S virus was equal to the level between the F mRNA with the HRS virus subgroup A virus and the subgroup B virus (Johnson and Collins, 1988, J. Gen. . Virol.
69: 2623-2628). The major open reading frame of the BRS virus F mRNA encodes a predicted protein of 574 amino acids, which was homologous to the size of the HRS virus F proteins (Collin
s et al., 1984, Proc. Natl. Acad. Sci. USA 81: 7683.
-7687; Baybutt and Pringle, 1987, J. Gen. Virol. 68:
2789-2796; Johnson and Collins, 1988, J. Gen. Virol.
69: 2623-2628; Lopez et al., 1988, Virus Res. 10: 249
-262). Features of the expected main structure of the F protein of BRS virus, N- terminal signal, the C- terminal anchor sequence, and is like a cleavage sequence for producing F ₁ and F ₂ polypeptides But these are HR
It was conserved with these features in the F protein of the S virus. The deduced amino acid sequence of the BRS virus F protein had 80% overall amino acid homology to the HRS virus F protein. F ₁ polypeptide of the BRS virus and HRS virus is stored more than F ₂ polypeptide having 68% amino acid homology, had a 88% amino acid homology. When the BRS virus and HRS virus and has been branched from a single common ancestor, low levels of amino acid homology of F ₂ compared to F ₁ in order to maintain a specific amino acid sequence, F ₁ peptide or different from the binding of the top, or less bound suggesting that might be present on F ₂ polypeptide. Moreover, when compared with the F ₁ polypeptide, differences in the level of homology between the F ₂ polypeptide with human and bovine, storage mechanism exact amino acid sequence of the F ₂ polypeptide, the F protein structure and higher storage mechanism of the amino acid sequence of F ₁ polypeptide in that it retains the function suggests that it is not critical. F ₁ polypeptide the amino acid sequence of the amino terminus of the proposed anchoring region and BRS viruses, when compared to these sequences in the F protein of HRS virus had been highly conserved. The proposed amino-terminal signal peptide of the F protein of the BRS and HRS viruses was not conserved in the amino acid sequence, but was conserved for its expected hydrophobic properties. The synthesis of the BRS virus F polypeptide in the presence of tikamycin, an inhibitor of N-linked glycosylation, results in BR
The S virus F polypeptide has been shown to be glycosylated by the addition of an N-linked carbohydrate moiety.
BRS synthesized in the presence of ticamycin
F ₂ polypeptide SDS virus and HRS virus
-Have the same electrophoretic mobility in polyacrylamide gels. This indicated that the F2 polypeptides of the BRS and HRS viruses differed in the degree of glycosylation. By nucleotide sequence analysis of the cDNA clones against the BRS virus F mRNA,
Differences in the number of possible glycosylation sites were demonstrated. Possible F ₂ amino acid sequence of the deduced BRS virus N-
While comprising only two sites for binding oligosaccharide addition, during F ₂ polypeptide of HRS virus A2 there are four possible sites. By utilizing tunicamycin to inhibit N-linked glycosylation, the HRS
Although differences in the electrophoretic mobility of the F ₁ polypeptide of virus and BRS virus was shown that not due to differences in glycosylation, F ₁ which is unglycosylated the BRS virus and HRS virus
This is because the electrophoretic mobilities of the polypeptides differ only slightly. Nucleotide sequence analysis, size and F ₁ polypeptide of F ₁ amino acid sequence and HRS virus BRS virus expected are the same, and one possible site for oligosaccharide addition of both of N- binding It was revealed to contain. Currently, BR
Slight differences present in the amino acid composition of the F ₁ polypeptide of S virus and HRS virus have concluded that the cause of the electrophoretic mobility differences. Changing a single amino acid in the G protein of vesicular stomatitis virus without altering the glycosylation of the protein,
A recent study demonstrating that electrophoretic mobility is altered supports this conclusion (Pitta et al., 198
9, J. Virol. 63: 3801-3809). The BRS virus F protein and the HRS virus F protein have conserved epitopes. Both convalescent bovine serum and monoclonal antibodies appear to recognize the F protein of either virus (Orvell et al., 198
7, J. Gen. Virol. 68: 3125-3135; Stott et al., 1984, D.
ev. Biol. Stand. 57: 237-244; Kennedy et al., 1988, J.
Gen. Virol. 69: 3023-3032; Lerch et al., 1989, J. Vi.
rol. 63: 833-840). Orvell et al. (1987, J. Gen. Vi
rol. 68: 3125-3135) found that only three out of 35 monoclonal antibodies raised against the F protein of the HRS virus did not recognize the F protein of three BRS virus strains. . Furthermore, all 11 monoclonal antibodies against the F protein have a neutralizing effect on the HRS virus, but they also
It also neutralized the infectivity of the S virus strain (Orvell et al., 19
87, J. Gen. Virol. 68: 3125-3135). In studies using synthetic peptides and monoclonal antibodies, HRS
It was suggested that at least two epitopes on the viral F protein were involved in virus neutralization. This epitope corresponds to amino acids 212 to 232 (Trud
el et al., 1987a, J. Gen. Virol. 68: 2273-2280; Trude
l et al., 1987b, Canad. J. Mirobiol. 33: 933-938),
And amino acids 283 to 299. The first of these epitopes is strictly conserved in the F protein of the BRS virus. In the second epitope, there are three changes in the F protein of the BRS virus, all of which are conservative changes. Although both of these epitopes are present on the F1 polypeptide, an amino acid that affects the neutralization was present concentrated in the F ₂ polypeptide on the fusion protein of Newcastle disease virus (Totoda
et al., 1988, J. Virol. 62: 4427-4430; Neyt et al., 19
89, J. Virol. 63: 952-954). HRS virus and BR
In contrast to the similarities of the F proteins of the S viruses, the G proteins of these viruses are antigenically distinct in nature. All monoclonal antibodies raised against the G protein of any HRS virus subgroup did not recognize the BRS virus G protein (Orvell et al., 1987, J. Gen. Virol. 68: 3125-313).
Five). It has been shown that polyclonal convalescent sera from cattle infected with the BRS virus recognized the G protein of the BRS virus but did not recognize the G protein of the HRS virus (Lerch et al., 1989, J. Vir.
ol. 63: 833-840). The differences in antigenic similarity between the FRS protein of the BRS virus and the HRS virus and the antigen cross-reactivity between the G protein of the BRS virus and the HRS virus also indicate the differences between the homologous proteins of the HRS virus and the BRS virus. It was reflected in the level of amino acid homology. Although the G proteins of the HRS and BRS viruses share only 30% amino acid homology, the F proteins of the two viruses have 80
% Amino acid homology. Differences in F and G glycoproteins are also significant in their epitope presentation. Garcia-Barreno et al. (1989, J.
Virol. 63: 925-932) can use a series of monoclonal antibodies to divide this F protein epitope into five non-overlapping groups, but many competitive response curves for epitopes on the G protein are quite overlapping. It was discovered that. The recombinant vaccinia virus containing the cDNA insert into the F gene of the BRS virus is
The protein was expressed. When the F protein of this BRS virus was initially cleaved into F ₁ and F ₂ polypeptides,
These had electrophoretic mobility on SDS-polyacrylamide gels similar to the F protein from cells infected with the BRS virus. In experiments with tunicamycin, an inhibitor of N-linked glycosylation,
The BRS virus F protein expressed from cells infected with the recombinant had been glycosylated to levels similar to the F protein from cells infected with the BRS virus. The F protein of the BRS virus expressed from the recombinant vaccinia virus is transported to and expressed on the surface of the infected cells as shown by surface immunofluorescence. 6. Microorganism Deposits The following microorganisms have been deposited with the American Type Culture Collection, Rockville, MD. Plasmid pRLF2012-76-1902 Deposit date: July 10, 1990 ATCC 40843 virus rVF-464 Deposit date: July 10, 1990 ATCCVR 2277 The present invention is disclosed in the microorganisms or the examples deposited. It is not limited in scope by the embodiments described, but these examples illustrate a few aspects of the invention, and any embodiments that are functionally equivalent thereto are within the scope of the invention. include. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to be included within the scope of the appended claims. Intended.
Numerous publications are cited herein, which are incorporated by reference in their entirety.

[Brief description of the drawings]

FIG. 1. cD of BRS virus F messenger RNA
NA sequencing strategy. The bottom scale is BRS virus F
Shows the number of nucleotides from the 5 'end of the mRNA.
The cDNA was inserted into M13mp19 replicative (RF) DNA and the nucleotide sequence was determined by dideoxynucleotide sequencing using M13-specific sequencing primers. The straight line for each clone indicates the portion of the mRNA sequence determined from that clone. Arrows indicate regions of F mRNA sequence determined by oligonucleotide extension on BRS virus mRNA. Oligonucleotides are BRS virus F mRNA
At bases 267-284. Clone F20, FB3
And the cDNA in FB5 was also cut out with PstI and KpnI, and M13mp1 was
It was inserted into 8RF-DNA. Clones F20, FB5 and
The cDNA fragment in FB3 was digested with the restriction enzyme EcoRI (FB
3, FB5), or AlwNI (F20), PflMI (F20) and Hpa
Generated by using I (F20) and EcoRV (F20), M1
The sequence of the internal region of F mRNA was determined by subcloning into 3mp19 or mp18 RF-DNA.

FIG. 2: Nucleotide sequence of BRS virus F mRNA. BRS virus FmR determined from cDNA clones and primer extension on BRS virus mRNA
1 shows the nucleotide sequence of NA. The dots above the sequence are placed every 10 nucleotides, and the number of the last base in each row is shown to the right of the sequence. The deduced amino acid sequences of the major open reading frames are also shown. The number of the last codon in each column is shown on the right side of the amino acid sequence. Possible N-linked glycosylation sites are boxed. The amino- and carboxy-terminal hydrophobic domains are underlined in black and are similar to the proposed amino-terminal hydrophobic regions of the F1 polypeptide. The proposed cleavage sequence is underlined in gray.

FIG. 3 is a hydrophilicity plot of F protein of BRS virus. The distribution of hydrophilic and hydrophobic regions along the deduced amino acid sequence of the F protein was determined using the algorithm of Kyte and Doolittle (1982, J. Mol. Biol. 157: 105-132). The value of each amino acid was calculated for a window of 9 amino acids. The bottom scale shows amino acid residues starting from the amino-terminal methionine. The hydrophilic region of the amino acid sequence is shown above the center line, and the hydrophobic region is shown below the center line.

FIG. 4. Sequence matching between the BRS virus F protein and the HRS virus A2, Long, RSS-2 and 18537 F proteins. Matching compares the BRS virus F protein against different HRS virus F proteins and
Leman and Wunsch method (1970, J. Mol. Biol. 48: 443
-453). Only non-identical amino acids are shown for the HRS virus F protein. The dots below the sequence are placed every 10 amino acids, and the number of the last residue in each row is shown to the right of the sequence. Possible N-linked glycosylation sites on the protein are boxed. 5
Cysteine residues conserved between all three proteins are indicated by triangles. The amino- and carboxy-terminal hydrophobic domains are underlined in black and are similar to the proposed amino-terminal hydrophobic regions of the F1 polypeptide. The proposed cleavage sequence is underlined in gray.

FIG. 5. Comparison of proteins from BRS virus and HRS virus infected cells synthesized in the presence and absence of tunicamycin. Protein from cells infected with BRS virus (B), HRS virus (H), or sham (M) was determined by the presence of tunicamycin (lanes B _T , H
_T and M _T ) or absent (lanes B, H and M)
Labeled below by [ ³⁵ S] methionine incorporation. Virus-specific proteins were immunoprecipitated using anti-RS virus serum and separated by electrophoresis on a 15% SDS-polyacrylamide gel. 3 shows an autoradiograph of a gel. All lanes are from the same gel and lane H
And H _T were exposed longer than the other lanes. [ ¹⁴ C] -labeled protein molecular weight markers are indicated in kilodaltons by their molecular weight.

FIG. 6. BR from recombinant vaccinia virus infected cells
Expression of S virus F protein. Recombinant vaccinia virus (moi = 10), wild-type vaccinia virus on BT cells
(moi = 10) or BRS virus (moi = 1). Proteins from recombinant and wild-type vaccinia virus (VV) containing the BRS virus F gene in the forward (lane F464 +) and reverse (F1597-) orientations are 3% of infectious.
-6 hours later, the cells were radiolabeled by [ ³⁵ S] methionine incorporation. Proteins from BRS virus infection (lane B) and mock infected cells (lane M) were also radiolabeled 24 hours after infection by incorporation of [ ³⁵ S] methionine for 3 hours. Proteins were immunoprecipitated using Wellcome anti-RS serum and compared by electrophoresis on a 15% polyacrylamide-SDS gel. Fluorography was performed on the gel and an autoradiograph is shown. The F, N, M and P proteins of the BRS virus are shown.
[ ¹⁴ C] -labeled protein molecular weight markers are indicated in kilodaltons by their molecular weight.

FIG. 7: BRS wis synthesized in the presence of tunicamycin
Russ-expressed F protein and recombinant vaccinia virus expression
Comparison of F proteins. Recombinant vaccinia wi in BT cells
Lus (moi = 10), wild-type vaccinia virus (moi = 10)
Alternatively, the cells were infected with the BRS virus (moi = 1). BRS
Recombinant virus containing the F gene of Irus in the forward direction (F464 = F)
Xinia virus, wild-type vaccinia virus (V),
BRS virus infection (B) and pseudo-infected cells (M)
These proteins are present at 3 hours of infection in vaccinia-infected cells.
24 hours after infection for BRS virus infected cells
In the absence of tunicamycin (lanes F, V, B, M)
Or presence (lane F_T , V _T , B_T , M_T )
³⁵Incorporate S] -methionine for 3 hours and radiolabel
Was. Protein is immunoprecipitated using Wellcome anti-RS serum
And run on a 15% polyacrylamide-SDS gel.
They were compared by electrophoresis. Perform fluorography on the gel
And an autoradiograph is shown. BRS virus
1 shows F, N, M and P proteins. [¹⁴C] -Sign
The molecular weight markers of proteins are determined by their molecular weight.
Shown in daltons.

FIG. 8: Surface immunofluorescence of HEp-2 cells infected with recombinant vaccinia virus. HEp grown on glass coverslips
-2 cells were simulated (M) infected, wild-type vaccinia virus (VV; moi = 10), recombinant virus F464 (rVVf;
oi = 10). 24 hours after infection, live cells are treated with anti-3
91-2 serum, then fluorescein-conjugated anti-bovine IgG (H +
L). The phase contrast (left panel) and fluorescence (right panel) micrographs are shown.

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[Procedure amendment]

[Submission date] February 5, 2001 (2001.2.5)

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] All figures

[Correction method] Change

[Correction contents]

FIG.

FIG. 2

FIG. 3

FIG. 5

FIG. 4

FIG. 6

FIG. 7

FIG. 8

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61P 37/04 C07K 16/10 C07K 14/115 C12N 1/15 16/10 1/19 C12N 1/15 1/21 1 / 19 7/00 1/21 C12P 21/02 C 5/10 21/08 7/00 C12Q 1/68 A C12P 21/02 1/70 21/08 G01N 33/15 Z C12Q 1/68 33/50 T 1/70 Z G01N 33/15 33/53 D 33/50 M 33/566 33/53 33/569 L 33/577 B 33/566 C12R 1:91) 33/569 (C12N 7/00 33/577 C12R 1:93) // (C12N 5/10 C12N 15/00 ZNAA C12R 1:91) A61K 37/02 (C12N 7/00 C12N 5/00 A C12R 1:93) C12R 1:91) (72) Inventor Larch, Robert United States 53711 Medison, Wisconsin, AP. 1, Charlet Gardens Court 2444

Claims (56)

[Claims] 1. A recombinant DNA molecule comprising a nucleic acid sequence encoding the bovine RS virus F protein. 2. The recombinant DNA molecule of claim 1, wherein the nucleic acid sequence or subsequence encoding the bovine RS virus F protein is substantially as shown in FIG. 2 and comprises at least about 10 nucleotides. 3. The nucleic acid sequence or subsequence encoding the bovine respiratory syncytial virus F protein is from about nucleotide 14 to about nucleotide 1735 substantially as shown in FIG. 2 and comprises at least about 10 nucleotides. Item 1
A recombinant DNA molecule as described. 4. The recombinant DNA molecule of claim 2, which is contained in plasmid pRLF2 012-76-1902 deposited with the ATCC under accession number 40842. 5. A sequence encoding a protein substantially homologous to the amino acid sequence shown in FIG.
A recombinant DNA molecule comprising a portion thereof comprising 6. The expression of a nucleic acid sequence encoding a bovine respiratory syncytial virus F protein or peptide fragment is regulated by a second nucleic acid sequence, and as a result, the recombinant DN
The recombinant DNA molecule according to claim 1, 2 or 5, wherein the bovine RS virus F protein is expressed in a host transformed with the A molecule. 7. The recombinant DN of claim 6, which is contained in rVF-464 deposited with the ATCC under accession number VR 2277.
A molecule. 8. The recombinant DNA according to claim 1, 2 or 5.
A recombinant microorganism containing the molecule. 9. The recombinant microorganism according to claim 8, which is a bacterium. 10. The recombinant microorganism according to claim 8, which is a yeast. 11. A recombinant microorganism comprising the recombinant DNA molecule according to claim 6. 12. The recombinant DN according to claim 1, 2 or 5.
A eukaryotic cell containing the A molecule. 13. A eukaryotic cell comprising the recombinant DNA molecule according to claim 6. 14. A eukaryotic cell comprising the recombinant DNA molecule according to claim 7. 15. A recombinant microorganism containing the DNA molecule according to claim 1, 2 or 5, is grown so that the DNA molecule is expressed by the microorganism, and the expressed bovine RS virus F protein or fragment is expressed. A method for producing a bovine RS virus F protein or a fragment thereof, the method comprising isolating. 16. Growing a recombinant microorganism containing the DNA molecule according to claim 6 so that the DNA molecule is expressed by the microorganism, and isolating the expressed bovine RS virus F protein or fragment. A method for producing a bovine RS virus F protein or a fragment thereof, comprising: 17. A bovine respiratory syncytial virus F protein or fragment expressed by growing a recombinant microorganism containing the DNA molecule according to claim 1, 2 or 5 so that the DNA molecule is expressed by a eukaryotic cell. A method for producing a bovine respiratory syncytial virus F protein or a fragment thereof. 18. A recombinant microorganism comprising the DNA molecule of claim 6 is grown so that said DNA molecule is expressed by a eukaryotic cell, and the expressed bovine RS virus F protein or fragment is isolated. A method for producing a bovine RS virus F protein or a fragment thereof. 19. A recombinant microorganism comprising the DNA molecule of claim 7 is grown so that the DNA molecule is expressed by a eukaryotic cell, and the expressed bovine RS virus F protein or fragment is isolated. A method for producing a bovine RS virus F protein or a fragment thereof. 20. The method according to claim 15, wherein the microorganism is a bacterium. 21. The method according to claim 16, wherein the microorganism is a bacterium. 22. The product of claim 15. 23. The product according to claim 16. 24. The product according to claim 17. 25. The product according to claim 18. 26. The product according to claim 19. 27. A subunit vaccine comprising the product of claim 15 in a suitable pharmaceutical carrier. 28. A subunit vaccine comprising the product of claim 16 in a suitable pharmaceutical carrier. 29. A subunit vaccine comprising the product of claim 17 in a suitable pharmaceutical carrier. 30. A subunit vaccine comprising the product of claim 18 in a suitable pharmaceutical carrier. 31. A subunit vaccine comprising the product of claim 19 in a suitable pharmaceutical carrier. 32. A subunit vaccine comprising the product of claim 20 in a suitable pharmaceutical carrier. 33. The infectious but non-pathogenic agent of claim 1,
A recombinant virus comprising the DNA molecule of 2 or 5. 34. A recombinant virus which is infectious but non-pathogenic and comprises the DNA molecule of claim 6. A vaccine comprising the recombinant virus according to claim 31. 36. A vaccine comprising the recombinant virus according to claim 34. 37. A recombinant or synthetic protein having an amino acid sequence substantially as shown in FIG. 4 or a partial sequence thereof comprising an antigenic determinant. 38. A subunit vaccine comprising the recombinant or synthetic protein according to claim 37 in a suitable pharmaceutical carrier. 39. An antibody produced by a method comprising inoculating an animal with the recombinant or synthetic protein according to claim 37. 40. The antibody according to claim 39, which is a monoclonal antibody. 41. An antibody fragment or antibody derivative produced from the antibody according to claim 39. 42. An antibody fragment or antibody derivative produced from the antibody according to claim 40. 43. A method for diagnosing bovine respiratory syncytial virus infection, comprising detecting the presence of bovine respiratory syncytial virus virus nucleic acid in a sample obtained from an animal likely to be infected with bovine respiratory syncytial virus. 44. (i) A method for preparing a nucleic acid prepared from a sample obtained from an animal likely to be infected with a bovine respiratory syncytial virus, wherein the nucleic acid has a sequence or subsequence substantially as shown in FIG. Contacting the nucleic acid molecule with the nucleic acid molecule under conditions permitting hybridization; and (ii) detecting whether hybridization has occurred, wherein the detection of hybridization is indicative of bovine respiratory syncytial virus infection. 44. The method of claim 43. 45. The method of claim 44, wherein RNA is prepared from an animal that is likely to be infected with the bovine respiratory syncytial virus and subjected to Northern blot analysis in step (i). 46. (i) A serum sample obtained from an animal which is likely to be infected with bovine respiratory syncytial virus is recombined with an amino acid sequence substantially as shown in FIG. Contacting the synthetic protein under conditions that allow binding of the antibody to the protein; and (ii) detecting whether binding of the antibody to the protein has occurred, wherein binding of the antibody to the protein is performed. A method for diagnosing bovine respiratory syncytial virus infection, wherein the method is indicative of exposure or infection of bovine respiratory syncytial virus. 47. The method according to claim 46, which is an enzyme-linked immunosorbent assay.
The described method. 48. The method of claim 46, comprising detecting the presence of the antibody by Western blot. 49. A method for eliciting an immune response against a bovine respiratory syncytial virus comprising administering to an animal in need of treatment an effective amount of the subunit vaccine of claim 27. 50. A method for eliciting an immune response against a bovine respiratory syncytial virus comprising administering to an animal in need of treatment an effective amount of the subunit vaccine of claim 28. 51. A method for eliciting an immune response against bovine respiratory syncytial virus comprising administering to an animal in need of the following treatment an effective amount of the subunit vaccine of claim 29. 52. A method for eliciting an immune response against a bovine respiratory syncytial virus comprising administering to an animal in need of treatment an effective amount of the subunit vaccine of claim 30. 53. A method of eliciting an immune response against bovine respiratory syncytial virus comprising administering to an animal in need of the following treatment an effective amount of the subunit vaccine of claim 31. 54. A method for eliciting an immune response against a bovine respiratory syncytial virus comprising administering to an animal in need of treatment an effective amount of the subunit vaccine of claim 32. 55. A method for eliciting an immune response against a bovine respiratory syncytial virus comprising administering to an animal in need of treatment an effective amount of the vaccine of claim 35. 56. A method for eliciting an immune response against a bovine respiratory syncytial virus comprising administering to an animal in need of treatment an effective amount of the vaccine of claim 36.